Disorders of iron metabolism result in morbidity and mortality for millions of individuals worldwide. Cellular uptake of iron by receptor mediated endocytosis of transferrin is well-characterized, but many aspects of iron transport are poorly understood. Red blood cells are highly dependent upon the transferrin cycle for iron acquisition, but post-transferrin cycle transport events are unknown. Molecular and cellular processes involved in intestinal iron absorption have not been elucidated, though uptake of dietary iron is clearly independent of the transferrin cycle. A unique mouse model offers critical insights into these pathways. Microcytosis (mk) is an autosomal recessive defect in murine iron acquisition which results in iron deficiency anemia, characterized by abnormalities in both intestinal and red cell iron uptake. Isolation of the gene defective in mk mice would be an important contribution to the understanding of mammalian iron metabolism. We propose to accomplish this by positional cloning, through linkage analysis of intercross and backcross progeny of inbred strains of mice segregating the mk trait and polymorphic DNA markers. A candidate genomic interval will be identified, and a genomic contig will be screened for genes expressed in red cells and intestine. Likely candidate genes will be analyzed for mutations specific to mk DNA. In parallel, we will generate immortalized erythroid cell lines from mk animals, to develop a functional screen for rescue of iron transport. These cell lines will be used for two purposes: to attempt to clone the mk gene directly by complementation, and to test candidate genes for restoration of activity.